1. Field of the Invention
The present invention relates to a method for fabricating a biosensor composed of a signal detector and a signal transducer. More particularly, the present invention relates to the direct immobilization of the signal detector on the signal transducer by use of hydrophilic polyurethane, thereby fabricating a biosensor which is superior in specificity, selectivity, and stability.
2. Description of the Prior Art
Targeting biochemical materials as analytes or as leads during signal generation, biosensors, like typical chemical sensors, consist mainly of signal detectors and signal transducers. In the signal detectors, bio-active reagents such as enzymes, antibodies, receptors, cells, etc., react with recognizable biochemical objects such as substrates, antigens, ligands, cells, etc., to cause physical and/or chemical signals which are quantitated in the signal transducers with the aid of physical or chemical measuring apparatus. For converting the physical or chemical signals into measurable ones, there may be taken an optical technique in which optical density, fluorescence or absorbance is measured, an electrochemical technique such as voltametry, potentiometry or conductometry, a calorimetric technique such as thermometry, or a mechanical change sensing technique such as a surface acoustic wave sensing method. Of them, electrochemical biosensors are now known to be suitable for the rapid and accurate determination of various metabolites, including urea, glucose, cholesterol, lactate, creatinine, amino acids, etc. Particularly, since the late 1970s in which glucose biosensors were developed to diagnose diabetes at home, biosensors have been applied for a wide range of fields; e.g., medical and diagnostic fields for drug detection, food and sanitary fields, environmental fields, and manufacturing process fields.
The most important factors for the analysis of biosensors include the specificity and the sensitivity for the targets to be analyzed. While the specificity for targets is dependent on the biochemical properties of the bio active reagents, the sensitivity is determined by the recognition efficiency of sensing elements and the performance of signal detection and signal transduction in the biosensors fabricated. Biosensors enjoy a characteristic advantage, different from those which chemical sensors have, of showing excellent specificity for targets as well as maintaining high sensitivity by virtue of the molecular recognition of the bio active reagents.
However, biosensors also suffer from a disadvantage in that it is very difficult to obtain highly reliable and accurate analysis results because the targets exist at trace amounts with great difference in biochemical properties and molecular weights and the signals are generated through complex reaction pathways. In order to maximize the sensitivity of biosensors with efficiency in the detection of analytes, the bio-active reagents are preferably immobilized in a layer adjacent to the sensing surface of the basic electrochemical transducer. More preferably, the bio-active reagents are immobilized in such a way to react with their targets at maximal magnitudes. Another requirement is that the bio-active reagents should maintain their entities without reduction or extinction of their biochemical activity. Therefore, the immobilization of the bio-active reagents, such as enzymes or functional proteins, on an appropriate support occupies an important position in the research on biosensors. In addition, active research has been directed to the introduction of the biosensors into solid-state electrodes because they can offer the advantage of miniaturization, and mass fabrication for cost reduction.
To approach the immobilization of the bio-active reagents, chemical techniques, physical techniques or combinations thereof are usually used. First, as for the physical techniques, they are representatively exemplified by the adsorption of the bio-active reagents in water-immiscible carriers and the entrapment of the bio-active reagents in water-immiscible polymer gels. The chemical immobilization is accomplished via, for example, covalent bonds or crosslinks.
In the adsorption method, which is the oldest in immobilizing the bio-active reagents in or on supports, advantage is taken of the hydrophilicity, hydrophobicity or ionic reciprocal reaction between the bio-active reagents, such as enzymes, antibodies, receptors, cells, etc., and the supports, such as membranes or films. The molecules immobilized through the adsorption are disadvantageous in that they are easily desorbed from the signal transducers.
Physically trapping the bio-active reagents in membranes or films, the entrapment method is routinely applied for where non-chemical treatments or mild reaction conditions are needed. This method is known to be most suitable for the labile bio-active materials which are liable to lose activity under a strong condition, but the bio-active reagents are loosely attached to the supports, so they are easily detached therefrom, thus decreasing the performance of the biosensors.
The covalent bonds through which the bio-active reagents are chemically immobilized on the surface of the signal transducers are typically achieved by the reaction with activated surface functional groups, such as peptide, thiol, amine and epoxy. The surface of the biosensors thus fabricated is stable to pH, ionic strength and temperature change. However, there appears a problem in that the molecules immobilized by covalent bonds, especially enzymes or receptors, may be restricted in activity.
Taking advantage of both the entrapment method and the covalent bond method, the crosslinking immobilization method uses a crosslinking agent to form additional chemical bonds through which the bio-active reagents are immobilized in membranes or films. In this regard, glutaraldehyde or hexamethylene diisocyanate serves as a crosslinking agent.
The most prevailing immobilization method is to use glutaraldehyde to bond enzymes to poly(vinyl chloride) (PVC). This immobilization method, however, suffers from disadvantages in that poor bonds exist between the enzyme layer and the hydrophobic polymer, resulting in deteriorating the performance and life span of the biosensors.
Recently, to solve the above problems, there have been developed an asymmetrically modified polymeric ion selective membrane on the surface of which a thin layer of enzyme can be formed for use in biosensors. The asymmetric membrane is based on polyurethane and prepared by coating a thin hydrophilic polyurethane film on a polyurethane film containing plasticizers and ionophores or ion carriers. In such an asymmetric membrane, a bio-active reagent is introduced on the surface of the hydrophilic polyurethane by the crosslinking method. Upon using glutaraldehyde as a crosslinking agent, an amine group is usually selected to afford a host in which an enzyme is anchored through a covalent bond, so as to form a thin layer of enzyme. The amine group may be provided to the hydrophilic polyurethane film by preparing it from a mixture of polylysine and hydrophilic polyurethane. Because the enzyme layer thus formed is found to be superb in the bonding strength to the ion-selective membrane, this is readily available to fabricate solid-state type biosensors. However, many complicated reaction steps and washing procedures which must be taken in the fabrication course of the biosensors make it difficult to commercialize the crosslinking method.
As a consequence of the intensive and thorough research on biosensors, repeated by the present inventors, it was found that a biosensor prepared by directly incorporating a sensing material into a hydrophilic polyurethane membrane shows superb stability and an exceptionally extended life span and the complicated process steps, such as those needed in conventional crosslinking methods, can be significantly reduced.
Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a method for fabricating a biosensor, which allows the simplification of the fabrication of biosensors without conducting complicated chemical reactions.
In accordance with the present invention, the above object could be accomplished by a provision of a method for fabricating a biosensor composed of a signal detector and a signal transducer, in which the signal detector is formed by mixing a sensing material with hydrophilic polyurethane and directly immobilizing the mixture on the signal transducer.